The biochemistry employed by bacteria differs markedly in many ways from that employed by humans. The enzyme Desll is an example that catalyzes an apparently radical-mediated deamination of TDP-4-amino-4,6-dideoxy-glucose. Desll employs S-adenosylmethionine (SAM) and a [4Fe-4S] cluster to effect this transformation strongly implicating it as a member of the radical SAM superfamily of enzymes. The mechanism of Desll is unique among these enzymes as it regenerates SAM during catalysis and does not require pyridoxal-5'-phosphate in order to make the target amino group labile, a characteristic of the aminomutases in the radical SAM superfamily. The goal of this proposal is to clarify the mechanistic details of the Desll catalyzed reaction, which has been proposed to proceed through either an anionic intermediate with direct elimination of the ammonia or a cationic intermediate with a carbinolamine product that eliminates ammonia non-enzymatically following dissociation. To achieve this objective, a detailed study of Desll kinetics will be undertaken with emphasis on pH dependence as well as kinetic isotope effects. The two hypotheses regarding the mechanism of Desll imply either an active site base or acid necessary for catalysis. The measurement of kinetic isotope effects will help to clarify the stepwise contributions to rate-limitation necessary for a complete characterization and interpretation of isotope effects probing ammonia elimination. The mechanistic alternatives also lead to different predictions regarding the fate of isotopically labeled atoms in the reactant as it is transformed into product. Characterization of products obtained by reacting deuterium or oxygen-18 labeled substrates with Desll can therefore test these hypotheses. Finally, enzyme intermediates cryotrapped during steady-state catalysis will be examined using electron paramagnetic resonance (EPR) spectroscopy. This will help to characterize the radical intermediates and clarify the Desll catalytic cycle. The Desll reaction is also an example of a bacterial deoxygenation reaction used to produce unusual sugars. These pathways are medically important, because the various products are implicated in both bacterial pathogenicity as well as antibiotic efficacy. Their characterization provides the foundation necessary for the development of effective antibiotics